Abstract
Several publications have documented the instability of transgene expression in plants. Previous genetic approaches to the study of transgene-associated phenotypes in plants were limited by this phenomenon. Here we show that a transgene can be expressed in plants with sufficient stability to allow an exhaustive mutagenic analysis of the resulting phenotype. We have expressed the morphogenic rolA gene from the TL-DNA of Agrobacterium rhizogenes Ri plasmid in transgenic Arabidopsis thaliana plants. The resulting pleiotropic RolA phenotype allows a visual screen for reversion to detect germinal as well as somatic instability of transgene expression. However no spontaneous reversions of the RolA phenotype were observed in 65 000 progeny of two independent transgenic A. thaliana lines, each carrying a single homozygous rolA locus. In contrast, 12 revertants of the RolA phenotype were isolated from 360000 ethyl methane sulphonate (EMS)-mutagenized M2 progeny. All revertants were shown genetically to carry stable recessive mutations in the rolA locus, thus establishing a series of loss-of-function alleles. Molecular characterization revealed that the loss-of-function alleles were structurally intact and expressed in all rolA mutants. A wild-type rolA locus and two loss-of-function alleles were reisolated and sequenced; base pair substitutions were found in each loss-of-function allele leading to single amino acid substitutions in the rolA open reading frame. Therefore no instability of expression of the rolA locus was detected in any of the 425 000 individuals studied in this analysis. Furthermore even under conditions of saturation mutagenesis, no extragenic suppressor locus was detected.
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References
Dehio C (1992) Isolierung und Charakterisierung von Suppressormutanten der Phänotypen rolA and rolB transgener Arabidopsis thaliana Pflanzen. Thesis, Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln, Köln, FRG
Deroles SC, Gardner RC (1988) Expression and inheritance of kanamycin resistance in a large number of transgenic petunias generated by Agrobacterium-mediated transformation. Plant Mol Biol 11:355–364
Dolferus R, Van Den Bossche D, Jacobs M (1990) Sequence analysis of two null-mutant alleles of the single Arabidopsis Adh locus. Mol Gen Genet 224:297–302
Gelvin SB, Karcher SJ, DiRita VJ (1983) Methylation of the T-DNA in Agrobacterium tumefaciens and in several crown gall tumours. Nucleic Acids Res 11:159–174
Hart MH, Fischer B, Neuhaus J-M, Meins F Jr (1992) Regulated inactivation of homologous gene expression in transgenic Nicotiana sylvestris plants containing a defense-related tobacco chitinase gene. Mol Gen Genet 235:179–188
Heberle-Bors E, Charvat B, Thompson D, Schernthaner JP, Barta A, Matzke AJM, Matzke MA (1988) Genetic analysis of T-DNA insertions into the tobacco genome. Plant Cell Rep 7:571–574
Hepburn AG, Clarke LE, Pearson L, White J (1983) The role of cytosine methylation in the control of nopaline synthase gene expression in a plant tumor. J Mol Appl Genet 2:315–329
Jorgensen R (1990) Altered gene expression in plants due to trans interactions between homologous genes. Trends Biotech 8:340–344
Kahl G, Weising K (1993) Genetic engineering of plant cells. In: Rehm H-J, Reed G (eds) Biotechnology. VCH Weinheim, pp 547–625
Kemper E, Grevelding C, Schell J, Masterson R (1992) Improved method for the transformation of Arabidopsis thaliana with chimeric dihydrofolate reductase constructs which confer methotrexate resistance. Plant Cell Rep 11:118–121
Kilby NJ, Leyser HMO, Furner IJ (1992) Promoter methylation and progressive transgene inactivation in Arabidopsis. Plant Mol Biol 20:103–112
Koncz C, Mayerhofer R, Koncz-Kalman Z, Nawrath C, Reiss B, Redei GP, Schell J (1990) Isolation of a gene encoding a novel chloroplast protein by T-DNA tagging in Arabidopsis thaliana. EMBO J 9:1337–1346
Linn F, Heidmann I, Saedler H, Meyer P (1990) Epigenic changes in the expression of the maize Al gene in Petunia hybrida: Role of numbers of integrated gnee copies and state of methylation. Mol Gen Genet 222:329–336
Matzke MA, Matzke AJM (1990) Gene interactions and epigenetic variation in transgenic plants. Dev Genet 11:214–223
Matzke MA, Primig M, Trnovsky J, Matzke AJM (1989) Reversible methylation of marker genes in sequentially transformed tobacco plants. EMBO J 8:643–649
Mayer U, Torres Ruiz RA, Berleth T, Miséra S, Jürgens G (1991) Mutations affecting the body organization in the Arabidopsis embryo. Nature 353:402–407
Meyer P, Linn F, Heidmann I, Meyer zA H, Niedenhof I, Saedler H (1992) Endogenous and environmental factors influence 35S promoter methylation of a maize Al gene construct in transgenic petunia and its colour phenotype. Mol Gen Genet 231:345–352
Meyerowitz EM (1989) Arabidopsis, a useful weed. Cell 56:263–269
Mittelsten Scheid O, Paszkowski J, Potrykus I (1991) Reversible inactivation of a transgene in Arabidopsis thaliana. Mol Gen Genet 228:104–112
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Otten L, De Greve H, Hernalsteens JP, Van Montagu M, Schieder O, Straub J, Schell J (1981) Mendelian transmission of genes introduced into plants by the Ti plasmids of Agrobacterium tumefaciens. Mol Gen Genet 183:209–313
Renckens S, De Greve H, Van Montagu M, Hernalsteens J-P (1992) Petunia plants escape from negative selection against a transgene by silencing the foreign DNA via methylation. Mol Gen Genet 233:53–64
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schmulling T, Schell J, Spena A (1988) Single genes from Agrobacterium rhizogenes influence plant development. EMBO J 7:2621–2629
Sinkar VP, Pythoud F, White FF, Nester EW, Gordon MP (1988a) RolA locus of the Ri plasmid directs developmental abnormalities in transgenic tobacco plants. Genes Dev 2:688–697
Sinkar VP, White FIT, Furner IJ, Abrahamsen M, Pythoud F, Gordon MP (1988b) Reversion of aberrant plants transformed with Agrobacterium rhizogenes is associated with the transcriptional inactivation of the TL-DNA genes. Plant Physiol 86:584–590
Slightom JL, Durand-Tardif M, Jouanin L, Tepfer D (1986) Nucleotide sequence analysis of TL-DNA of Agrobacterium rhizogenes agropine type plasmid. J Biol Chem 261:108–121
Spena A, Schmülling T, Koncz C, Schell J (1987) Independent and synergistic activity of rolA, B, and C loci in stimulating abnormal growth in plants. EMBO J 6:3891–3899
Valvekens D, Van Montagu M, Van Lijsebettens M (1988) Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc Natl Acad Sci USA 85:5536–5540
Walter C, Broer I, Hillemann D, Pühler A (1992) High frequency, heat treatment-induced inactivation of the phophinothricin resistance gene in transgenic single cell suspension cultures of Medicago sativa. Mol Gen Genet 235:189–196
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Dehio, C., Schell, J. Stable expression of a single-copy rolA gene in transgenic Arabidopsis thaliana plants allows an exhaustive mutagenic analysis of the transgene-associated phenotype. Molec. Gen. Genet. 241, 359–366 (1993). https://doi.org/10.1007/BF00284689
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DOI: https://doi.org/10.1007/BF00284689